In this proposal we aim to define the molecular mechanisms by which membrane-bound organelles are organized in magnetotactic bacteria. These microorganisms are capable of using the earth's magnetic field as a guide to simplify their search for low oxygen environments with the help of a specialized organelle termed the magnetosome. Magnetosomes are small lipid-bilayer invagination of the inner cell membrane within which nanometer-sized iron-based magnetic particles are produced. Individual magnetosomes are arranged into a chain that maximizes the interaction of the bacterium with magnetic fields. The work of many groups, including ours, has pinpointed a large number of genes as having specific roles in building the magnetosome organelle. One of these factors, MamK, is a bacterial actin-like protein that is at the center of magnetosome chain formation. In the absence of MamK, magnetosomes fail to form coherent chains and are instead separated by numerous gaps. Bacterial actins are widespread amongst the Bacteria and form numerous families with distinct functions. For instance, MreB is involved in directing the synthesis of the cell wall whereas ParM and other actins help in segregation of naturally occurring plasmids. Outside of MreB and ParM, little is known regarding the function and behavior of the vast majority of the bacterial actin-like proteins. MamK is the founding member of one of these families and its representatives are mostly found within the MB. With the support of this grant over the last four years, we have developed an in vitro system to study the polymerization kinetics of MamK and, through collaborations, obtained a high-resolution electron microscopic structure of its filaments. Furthermore, we identified two proteins, MamJ and LimJ that are responsible for the dynamic turnover of MamK filaments in vivo. Using a directed mutagenesis strategy, we have also identified intrinsic features of MamK that contribute to its function, localization, bundling and dynamics. Finally, we have used broader genetic strategies to define the functions of over 20 genes in the formation of magnetosomes and biomineralization of magnetic particles. In this proposal, we have will build on our exclusive expertise and infrastructure to define the mechanisms of MamK function and magnetosome formation in more detail. First, we will ask if the biochemical and structural features of MamK are represented in the diverse members of its family, some of which exist in non-magnetotactic bacteria as well as the archaea. Second, we will define and characterize regulators and interactors of MamK. Third, we will develop tools to ask if MamK is involved in chain establishment, maintenance or segregation. Finally, we will develop tools to image and understand the earliest steps of magnetosome membrane biogenesis. These experiments promise to shed mechanistic light on the cell biology of bacterial organelles and the evolution and functional diversity of bacterial actins.